Transgenic plant-based vaccines

ABSTRACT

A method of inducing partial or complete immunity to an infectious disease in a mammal comprising providing to the mammal for oral consumption an effective amount of a protein complex comprising five monomeric fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 10/920,648, titled “Methods and Substances forPreventing and Treating Autoimmune Disease,” filed Aug. 17, 2004, nowabandoned, which is a continuation of U.S. patent application Ser. No.09/296,981, titled “Methods and Substances for Preventing and TreatingAutoimmune Disease,” filed Apr. 22, 1999, now U.S. Pat. No. 6,777,546,issued on Aug. 17, 2004, which: 1) claims the benefit of U.S. PatentApplication No. 60/082,688, titled “Plant Vaccines Against AutoimmuneDisease,” filed Apr. 22, 1998 and 2) is a continuation-in-part of U.S.patent application Ser. No. 09/167,493, titled “Expression of CholeraToxin B Subunit in Transgenic Plants and Efficacy Thereof in OralVaccines,” filed Oct. 7, 1998, now abandoned, which claims the benefitof U.S. Patent Application No. 60/061,265, titled “Cholera Toxin in FoodPlants,” filed Oct. 7, 1997; and the present application is acontinuation of U.S. patent application Ser. No. 11/001,153, titled“Transgenic Plant-based Vaccines,” filed Nov. 30, 2004, now abandoned,which is a continuation of U.S. patent application Ser. No. 10/245,749,titled “Transgenic Plant-based Vaccines,” filed Sep. 16, 2002,nowabandoned , which is a divisional of U.S. patent application Ser. No.09/771,536, filed Jan. 29, 2001 now abandoned, which claims the benefitof U.S. Patent Application 60/178,403, titled “Production of a CholeraToxin B Subunit-rotavirus NSP4 Enterotoxin Fusion Protein in Potato,”filed Jan. 27, 2000; the contents of which are incorporated by referenceherein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support undersubcontract number 010-FY97-LLU-LANGRIDGE with the National Medical TestBed, United States Department of the Army. The United States Governmenthas certain rights in this invention.

BACKGROUND

Acute infectious enteric diseases, such as acute gastroenteritis, aresecond only to acute respiratory diseases as a cause of human deathworldwide. Cholera, rotavirus and enterotoxigenic E. coli are the threemajor causative agents of acute gastroenteritis. Human rotavirus, forexample, is the most important cause of infantile gastroenteritisworldwide. This virus has a tremendous public health impact worldwide,infecting nearly every child in the first few years of life. Rotavirusinfection is responsible for approximately 1 million deaths each yearand an estimated 18 million hospitalizations. 20% to 40% of thehospitalizations are for childhood diarrhea, which makes the rotavirusthe most important single cause of diarrheal mortality among children.

Treatment for acute gastroenteritis includes antibiotics and metabolicsupport. However, adequate treatment is often not available,particularly in lesser developed areas where the incidence of acutegastroenteritis is highest. Prevention of acute gastroenteritis would bepreferable to treatment. However, preventative measures, such as theprovision of safe drinking water, are often inadequate or unavailable.

Therefore, it would be useful to have a new method for the prevention ofacute gastroenteritis. Further, it would be particularly useful to havea method for the prevention of acute gastroenteritis which would preventmultiple types of acute gastroenteritis simultaneously.

SUMMARY

According to one embodiment of the present invention, there is provideda DNA construct that encodes, upon expression in a plant cell, a fusionprotein comprising a multimeric cholera toxin B subunit and a firstimmunogenic antigen from a causal factor of a first mammalian disease.The first immunogenic antigen can be a rotavirus antigen. The firstimmunogenic antigen can also be an enterotoxigenic E. coli antigen.

The fusion protein encoded by the DNA construct can further comprise asecond cholera toxin subunit. The second cholera toxin subunit can becholera toxin A2 subunit.

The fusion protein encoded by the DNA construct can further comprise asecond immunogenic antigen from a causal factor of a second mammaliandisease. The second immunogenic antigen can be a rotavirus antigen. Thesecond immunogenic antigen can also be an enterotoxigenic E. coliantigen. Either the first mammalian disease or the second mammaliandisease or both can be an infectious enteric disease.

According to another embodiment of the present invention, there isprovided a DNA construct that encodes, upon expression in a plant cell,a fusion protein comprising a cholera toxin A2 subunit, a multimericcholera toxin B subunit, a first immunogenic antigen from a causalfactor of a first mammalian disease, and a second immunogenic antigenfrom a causal factor of a second mammalian disease. The firstimmunogenic antigen can be a rotavirus antigen. The second immunogenicantigen can be an enterotoxigenic E. coli antigen. Either the firstmammalian disease or the second mammalian disease or both can be aninfectious enteric disease.

According to another embodiment of the present invention, there isprovided an expression vector comprising a DNA construct of the presentinvention, a transgenic plant cell transformed with a DNA construct ofthe present invention, a transgenic plant seed transformed with the DNAconstruct of the present invention, and a transgenic plant transformedwith the DNA construct of the present invention.

According to yet another embodiment of the present invention, there isprovided a method of producing an immunogen in a plant comprisingcultivating a transgenic plant of the present invention under conditionseffective to express the fusion protein.

According to another embodiment of the present invention, there isprovided a method of inducing partial or complete immunity to aninfectious disease in a mammal comprising providing to the mammal fororal consumption an effective amount of a plant of the presentinvention.

The present invention also includes means for producing, in a plantcell, a fusion protein comprising a multimeric cholera toxin B subunitand a first immunogenic antigen from a causal factor of a firstmammalian disease. The means can comprise a DNA construct that encodes,upon expression in the plant cell, a multimeric cholera toxin B subunitand a first immunogenic antigen from a causal factor of a firstmammalian disease. The first immunogenic antigen can be a rotavirusantigen. The first immunogenic antigen can also be an enterotoxigenic E.coli antigen. The fusion protein can further comprise a second choleratoxin subunit, such as cholera toxin A2 subunit. The fusion protein canfurther comprise a second immuhogenic antigen from a causal factor of asecond mammalian disease. The second immunogenic antigen can be arotavirus antigen. The second immunogenic antigen can also be anenterotoxigenic E. coli antigen.

The present invention also includes means for producing, in a plantcell, a fusion protein comprising a cholera toxin A2 subunit, amultimeric cholera toxin B subunit, a first immunogenic antigen from acausal factor of a first mammalian disease, and a second immunogenicantigen from a causal factor of a second mammalian disease. The firstimmunogenic antigen can be a rotavirus antigen. The second immunogenicantigen can be an enterotoxigenic E. coli antigen.

According to another embodiment of the present invention, there isprovided an expression vector comprising the means of the presentinvention, a transgenic plant cell transformed with means of the presentinvention, a transgenic plant seed transformed with the means of thepresent invention, and a transgenic plant transformed with the means ofthe present invention.

The present invention also includes a method of producing an immunogenin a plant comprising cultivating the transgenic plant of the presentinvention under conditions effective to express the fusion protein. Thepresent invention further includes a method of inducing partial orcomplete immunity to an infectious disease in a mammal comprisingproviding to the mammal for oral consumption an effective amount of aplant of the present invention.

According to another embodiment of the present invention, there isprovided a fusion protein comprising a multimeric cholera toxin Bsubunit and a first immunogenic antigen from a causal factor of amammalian disease. The first immunogenic antigen can be a rotavirusantigen. The first immunogenic antigen can also be an enterotoxigenic E.coli antigen. The fusion protein can further comprise a second choleratoxin subunit. The second cholera toxin subunit can be cholera toxin A2subunit. The fusion protein can further comprise a second immunogenicantigen from a causal factor of a second mammalian disease. The secondimmunogenic antigen can be a rotavirus antigen. The second immunogenicantigen can also be an enterotoxigenic E. coli antigen. Either the firstmammalian disease or the second mammalian disease or both can be aninfectious enteric disease.

In one embodiment, the fusion protein comprises a cholera toxin A2subunit, a multimeric cholera toxin B subunit, a first immunogenicantigen from a causal factor of a mammalian disease, and a secondimmunogenic antigen from a causal factor of a second mammalian disease.

According to another embodiment of the present invention, there isprovided a fusion protein encoded by the DNA construct of the presentinvention.

According to another embodiment of the present invention, there isprovided a method of inducing partial or complete immunity to aninfectious disease in a mammal comprising providing to the mammal fororal consumption an effective amount of the fusion protein of thepresent invention.

FIGURES

The features, aspects and advantages of the present invention willbecome better understood with regard to the following description,appended claims and accompanying figures where:

FIG. 1 is a diagram of the vector pPCV701FM4-CTB:NSP4; and

FIG. 2 is a diagram of the vector pPCV701CFA/I-CTB-NSP4.

DESCRIPTION

According to one embodiment of the present invention, there is provideda method of inducing partial or complete immunity to an infectiousdisease, such as gastroenteritis, in a mammal by administering to themammal a portion of a transgenic plant comprising a fusion protein,where the fusion protein comprises at least one cholera toxin subunitand an immunogenic antigen from a causal factor of the disease. In apreferred embodiment, the fusion protein comprises at least two choleratoxin subunits, at least one of which functions as an antigen, inaddition to functioning as an adjuvant for the immunogenic antigen. Inanother preferred embodiment, the fusion protein comprises at least twoimmunogenic antigens, each fused to a cholera toxin subunit. By fusingthe immunogenic antigen to the cholera toxin subunit, the fusion proteinmore specifically targets the appropriate immune system tissue uponadministration. This increased specificity compensates for the low levelof production of the protein in the transgenic plant and increases theresponse of the mammal's immune system.

In one embodiment, the fusion protein comprises the twenty-two aminoacid immunodominant epitope of the murine rotavirus enterotoxin NSP4fused to the cholera toxin B subunit (CTB). In another embodiment, thefusion protein comprises the enterotoxigenic E. coli (ETEC) fimbrialcolonization factor CFA/I fused to the cholera toxin A2 subunit (CTA2).In yet another embodiment, the fusion protein comprises both thetwenty-two amino acid immunodominant epitope of the murine rotavirusenterotoxin NSP4 fused to the cholera toxin B subunit, and the fusionprotein comprises the enterotoxigenic E. coli fimbrial colonizationfactor CFA/I fused to the cholera toxin A2 subunit.

Though the method is described in the context of preventinggastroenteritis by way of example, it will be understood by those withskill in the art with reference to this disclosure, that the presentmethod can be used to prevent other enteric infectious diseases andother non-enteric infectious diseases such as respiratory diseases. Themethod will now be described in more detail.

-   1) Construction of a Transgenic Plant Producing a Fusion Protein    Comprising the Immunodominant Epitope of the Murine Rotavirus    Enterotoxin NSP4 Fused to the Cholera Toxin B Subunit and    Confirmation of Transformation.

According to one embodiment of the present invention, there is provideda transgenic plant producing a fusion protein comprising the twenty-twoamino acid immunodominant epitope of the murine rotavirus enterotoxinNSP4 fused to the cholera toxin B subunit. The transgenic plant can beadministered to a mammal to immunize the mammal against cholera androtavirus infection simultaneously.

Referring now to FIG. 1, there is shown a diagram of the vector used toprepare the transgenic plant. As can be seen, the vector contained fourgenes located within the transferred DNA (T-DNA) sequence flanked by theright and left border (RB and LB), and 25 bp direct repeats of theborders required for integration of the transferred DNA into plantgenomic DNA. The four genes were the CTBH:NSP4(114-135):SEKDEL codingsequence under control of the mas P2 promoter; the bacterial luciferaseAB fusion gene (luxF) under control of the mas P1 promoter used as adetectable marker; an NPT II expression cassette used for resistance tokanamycin in plants; and a β-lactamase cassette for resistance toampicillin in E. coli and carbenicillin in A. tumefaciens. The g7pApolyadenylation signal was from the A. tumefaciens T_(L)-DNA gene 7. TheOcspA polyadenylation signal is from the octopine synthase gene. Pnoswas the promoter of the nopaline synthase gene g4pA was thepolyadenylation signal from T_(L)-DNA gene 4. OriT was the origin oftransfer derived from pRK2. OriV was the wide host range origin ofreplication for multiplication of the plasmid in A. tumefaciens derivedfrom pRK2. Ori pBR322 was the replication origin of pBR322 formaintenance of the plasmid in E. coli.

The vector pPCV701FM4-CTB:NSP4 was constructed as follows. The plantexpression vector pPCV701FM4, a derivative of plasmid pPCV701, wasdigested with XbaI and SacI restriction endonucleases within themultiple cloning site to insert a gene encoding the cholera toxin Bsubunit and its leader sequence, SEQ ID NO:1, from plasmid pRT42containing the ctxAB operon. The oligonucleotide 5′ primer(5′-gctctagagccaccatgattaaattaaaatttggtg-3′), SEQ ID NO:2, and the 3′primer (5′-ctggagctcgggccccggcccatttgccatactaattgcgg-3′), SEQ ID NO:3,were synthesized with XbaI and SacI restriction endonuclease recognitionsites (bold) for amplification and cloning of the CTB-hinge codingsequence, SEQ ID NO:4, in a model 394 DNA/RNA Synthesizer (AppliedBiosystems, Inc. Foster City, Calif. US)

The oligonucleotide sequence surrounding the translation initiationcodon of the CTB gene, SEQ ID NO:1, was altered to a preferrednucleotide context for translation in eukaryotic cells, (5′-gccacc-3′)and a putative Shine-Dalgarno sequence (AGGA) present in the ctxABoperon in plasmid pPT42 was removed. The DNA sequence, SEQ ID NO:5,encoding the 21 amino acid leader peptide of the CTB was retained todirect the nascent CTB fusion peptide into the lumen of the ER.

The 3′ primer, SEQ ID NO:3, was designed to contain a nucleotidesequence encoding a Gly-Pro box (Gly-Pro-Gly-Pro) with relatively lessfrequently used codons in plants to allow the ribosomes to halt forproper folding of CTB moiety before translation of the downstreammessage sequence. An additional function of the Gly-Pro box was to actas a flexible hinge between CTB and the conjugated peptide.

The methods for cloning the CTBH fusion gene, SEQ ID NO:4, into themultiple cloning site immediately downstream of the mas P₂ promoter andthe DNA sequence confirmation were as follows. PCR amplification wasperformed using a Gene Amp PCR System 9600, (The Perkin ElmerCorporation, Norwalk, Conn. US) according to the following reactionparameters; 94° C., 45 sec.: 55° C. for 60 sec.: 72° C. for 45 sec., 30cycles total. The ligated vector and PCRed fragment (T4 ligase at 16° C.for 20 hrs.) were electroporated into Escherichia coli strain HB101 (250μFD, 200Ω, and 2,500 volts; BioRad® Gene Pulser II unit (Bio RadLaboratories, Inc., Hercules, Calif. US) and ampicillin resistantcolonies were isolated after overnight growth at 37° C.

To confirm the presence of the correct CTBH fusion gene sequence, SEQ IDNO:4, in transformed E. coli cells, the plasmid was isolated fromindividual colonies of transformants and subjected to DNA sequenceanalysis with the forward primer (5′- accaatacattacactagcatctg-3′), SEQID NO:6, specific for the mas P₂ promoter and the reverse primer(5′-gactgagtgcgatattatgtgtaatac-3′), SEQ ID NO:7, specific for the gene7 poly(A) signal (model 373A DNA Sequencer, Applied Biosystems, Inc.).This plant transformation vector was designated as pPCV701FM4-CTBH.

To insert the rotavirus enterotoxin NSP4(114-135) epitope gene, SEQ IDNO:8, two overlapping primer sequences were synthesized and equimolaramounts of both single-stranded deoxyribonucleotide fragments weresubjected to PCR amplification (94° C. 45 sec.: 55° C. for 60 sec.: 72°C. for 60 sec.: 30 cycles total) to create double stranded 103 bp lengthsynthetic gene. The 5′ oligonucleotide, SEQ IDNO:9,5′-gccgagctcgataagttgactactagggagattgagcaagttgagttgttgaagaggatt-3′and the 3′ oligonucleotide, SEQ ID NO:10,5′-gccgagctcacaactcatccttctcagaagtcaacttatcgtaaatcctcttcaacaact-3′ weredesigned to contain 17 bp complementary sequence for the thermostableVent DNA polymerase (New England Biolabs, Beverly, Mass. US) attachmentsite for the initial cycle of the PCR reaction. The 3′ oligonucletide,SEQ ID NO:10, contained the DNA sequence encoding endoplasmic reticulumretention signal (SEKDEL) with codons most frequently found in potatoplants. Both oligonucleotides contains SacI recognition sites (bold) toclone the synthetic gene fragment into SacI site immediately downstreamof the hinge sequence of the vector to create vectorpPCV701FM4-CTBH:NSP4.

Following confirmation of the correct fusion gene sequence,CTBH:NSP4(114-135):SEKDEL, SEQ ID NO:11, the shuttle vector wastransferred into A. tumefaciens recipient strain GV3101 pMP90RK by thesame electroporation conditions used for E. coli transformation. A.tumefaciens transformants were grown at 29° C. on YEB solid mediumcontaining the antibiotics carbenicillin (100 μg/ml), rifampicin (100μg/ml), kanamycin (25 μg/ml), and gentamycin (25 μg/ml) for selection oftransformants.

The plasmid was isolated from an A. tumefaciens transformant andtransferred back into E. coli HB101 by electroporation, and restrictionendonuclease analysis was used to confirm that no significant deletionhad occurred in the vector. Structural confirmation of the plasmid wasrequired because recombination events within the rec⁺ A. tumefaciensstrain could alter the T-DNA sequence. Transfer of the plasmid from A.tumefaciens back to the E. coli host was necessary because significantamounts of plasmid are difficult to isolate directly from A.tumefaciens. Agrobacteria carrying the plant expression vector weregrown on YEB solid medium containing all four antibiotics for 48 hoursat 29° C. and directly used for transformation of sterile potato leafexplants.

Sterile potato plants S. tuberosum cv. Bintje were grown in Magentaboxes (Sigma Chemical Co., St. Louis, Mo. US) on solid Murashige andSkoog (MS) complete organic medium (JRH Biosciences, Lenexa, Kans. US)containing 3.0% sucrose and 0.2% gelrite. Leaf explants excised from theyoung plants were laterally bisected in a 9 cm diameter culture dishcontaining an overnight culture of A. tumefaciens suspension (1×10¹⁰cell/ml) harboring pPCV701FM4-CTBH:NSP4. The bacterial suspension wassupplemented with acetosyringone (370 μM) to increase transformationefficiency. The explants were incubated in the bacterial suspension for5 minutes, blotted on sterile filter paper, and transferred to MS solidmedium, pH 5.7, containing 0.1 μg/ml naphthalene acetic acid (NAA) and1.0 μg/ml trans-zeatin. The leaf explants were then incubated for 48hours at room temperature on MS solid medium to permit T-DNA transferinto the plant genome. For selection of transformed plant cells and forcounter selection against continued Agrobacterium growth, the leafexplants were transferred to MS solid medium containing the antibioticskanamycin (100 μg/ml) and claforan (300 μg/ml).

Transformed plant cells formed calli on the selective medium aftercontinuous incubation for 2 to 3 weeks at room temperature in a lightroom under cool white fluorescent tubes on a 12 hour photoperiod regime.When transformed calli grew to between 5 mm and 10 mm in diameter, theleaf tissue was transferred to MS medium containing 1.0 μg/mltrans-zeatin, 50 μg/ml kanamycin and 400 μg/ml claforan for shootinduction. Regenerated shoots were excised and transferred to MS solidmedium without plant hormones or antibiotics to stimulate rootformation. Plantlets were allowed to grow and form microtubers understerile conditions to characterization.

Luciferase activity was detected in transformed A. tumefaciens andtransgenic plants as follows. The presence of the plant expressionplasmid in agrobacteria, luxF gene expression under control of the masP1 promoter was monitored by low-light image analysis. To perform thebioluminescent assay, bacterial culture grown for 24 hours on YEB solidculture medium was covered with a glass culture plate lid swabbed withsubstrate n-decyl aldehyde and analyzed by the Argus-100 intensifiedcamera system (Hamamatsu Photonics UK Ltd., Bridgewater, N.J. US).

Expression of luxF gene was also monitored to confirm the presence ofthe CTBH:NSP4(114-135):SEKDEL, SEQ ID NO:11, in the plant genome and toestimate the level of CTB fusion gene expression by mas P2 promoter.Leaves excised from putative transformants were wounded by scalpel bladefollowed by incubation on MS solid medium containing naphthalene aceticacid (5 μg/ml) and 2,4-dichlorophenoxy acetic acid (6 μg/ml) for 48hours. Light emission from the wounded leaf tissues was detected asdescribed for agrobacteria.

More than forty independent kanamycin-resistant plants were regeneratedfrom Agrobacterium mediated transformation of potato leaf explants withthe plant expression vector pPCV701FM4-CTBH:NSP4. Three of the fortyplants were found to express luciferase activities above backgroundlevels from untransformed plants. No luciferase activity was detected inleaves of untransformed potato plants.

The three transformed potato plants showing luciferase activities wereanalyzed for the presence of the fusion gene in plant genomic DNAisolated from young leaf tissues as follows. Genomic DNA was isolatedfrom the transformed potato leaf tissues. Presence of the CTB fusiongene was determined by PCR analysis using the oligonucleotide primersspecific for the T-DNA sequence. Transformed plant genomic DNA (500 ng)was used as a template to detect the CTB gene by PCR amplification (94°C. for 45 sec.: 55° C. for 60 sec.: 62° C. for 60 sec. for a total of 30cycles). A 650 bp DNA fragment including both 5′ and 3′ flankingsequences of the fusion gene, was amplified. The PCR amplification wasvery specific probably due to high specificity of the primers used forthe PCR reaction.

The DNA fragments amplified from plasmid vector pPCV701FM4-CTBH:NSP4 andfrom transgenic plant genomic DNA were identical in molecular weight.Although, identical amounts of template genomic DNA (500 ng) was usedfor the PCR reaction, the plant exhibiting the highest luciferaseactivity also demonstrated the highest level of PCR amplification.

The presence of the CTB fusion protein was detected in transformedpotato tissues as follows. Transgenic potato leaf and microtuber tissueswere analyzed for the CTB fusion gene expression by immunoblot analysis.Callus tissues were derived from leaf or tuber tissues incubated for 4weeks on MS solid medium containing 5.0 mg/l NAA and 6.0 mg/l 2,4-D.Tissues were homogenized by grinding by a mortar and pestle at 4° C. inextraction buffer (1:1 w/v) (200 mM Tris-Cl, pH 8.0, 100 mM NaCl, 400 mMsucrose, 10 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 0.05% Tween-20). The tissue homogenate was centrifuged at17,000×g in a Beckman GS-15R centrifuge for 15 minutes at 4° C. toremove insoluble cell debris. An aliquot of supernatant containing 100μg of total soluble protein, as determined by Bradford protein assay(Bio Rad Laboratories, Inc.), was separated by 15% sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) at 125 volts for 30 to 45minutes in Tris-glycine buffer (25 mM Tris, 250 mM glycine, pH 8.3, 0.1%SDS). Samples were either loaded directly on the gel or boiled for 5minutes prior to electrophoresis.

The separated protein bands were transferred from the gel toapproximately 80 cm² Immun-Lite membranes (Bio Rad Laboratories, Inc.)by electroblotting on a semi-dry blotter (Labconco, Kansas City, Mo. US)for 60 minutes at 15 V and 100 mA. Nonspecific antibody reactions wereblocked by incubation of the membrane in 25 ml of 5% non-fat dry milk inTBS buffer (20 mM Tris pH 7.5 and 500 mM NaCl) for 1 hour with gentleagitation on a rotary shaker (40 rpm), followed by washing in TBS bufferfor 5 minutes. The membrane was incubated overnight at room temperaturewith gentle agitation in a 1:5,000 dilution of rabbit anti-choleraantiserum (Sigma C-3062) in TTBS antibody dilution buffer (TBS with0.05% Tween-20 and 1% non-fat dry milk) followed by washing three timesin TBST washing buffer (TBS with 0.05% Tween-20). The membrane wasincubated for 1 hour at room temperature with gentle agitation in a1:10,000 dilution of mouse anti-rabbit IgG conjugated with alkalinephosphatase (Sigma A-2556) in antibody dilution buffer. The membrane waswashed three times in TTBS buffer as before and once with TBS buffer,followed by incubation in 1× chemiluminescent substrate CSPD™ (Bio RadLaboratories, Inc.) for 5 minutes at room temperature with gentleagitation. The membrane was then wrapped with transparent plasticmembrane and placed in a photocassette on Kodak X-OMAT film (cat#1651454). (The membrane was also used to image chemiluminescent lightintensity in both the numerical and graphic form by the Argus-100 videoimage analysis.) The film was subjected to 1-10 minutes exposure anddeveloped in a Kodak M35A X-OMAT Processor.

Using this method, transgenic potato tuber tissues from the threetransformed plants were shown to contain CTB fusion protein (˜60 kDa)that strongly reacted with anti-cholera toxin antibody whichpredominantly recognized pentameric form of cholera toxin or its Bsubunit. Potato plants transformed with the plant expression vectoronly, which did not contain the CTBH:NSP4(114-135):SEKDEL sequence, SEQID NO:11, did not show this protein band. One plant, designated Plant#1, showed approximately 3 to 5 fold higher chimeric protein level thanthe other two plants, Plant #2 and Plant #3. Potato-synthesized CTB-NSP4fusion peptide exhibited higher molecular weight than both pentamericbacterial CTB subunit (45 kDa) and potato-synthesized pentameric CTBsubunit with ER retention signal (50 kDa).

The level of CTB fusion protein in the in tubers of transgenic Plant #1was quantified using both chemiluminescent G_(M1)-ELISA andchemiluminescent immunoblot assays as follows. Pentameric CTB fusionprotein levels in transgenic potato plants and its affinity forG_(M1)-ganglioside were evaluated by quantitative chemiluminescentG_(M1)-ELISA assays. The microtiter plate was coated with 100 μl/well ofmonosialoganglioside G_(M1) (3.0 μg/ml) (Sigma G-7641) in bicarbonatebuffer, pH 9.6 (15 mM Na₂CO₃, 35 mM NaHCO₃) and incubated at 4° C.overnight. Wells were loaded with 100 μl/well of 10-fold serialdilutions of total soluble potato leaf or tuber protein in phosphatebuffered saline (PBS) and incubated overnight at 4° C. The plate waswashed three times in PBST (PBS containing 0.05% Tween-20). The wellswere blocked by adding 300 μl/well of 1% bovine serum albumin (BSA) inPBS and incubated at 37° C. for 2 hours followed by washing three timeswith PBST. The wells were loaded with 100 μl/well of 1:5,000 dilution ofrabbit anti-cholera toxin antibody (Sigma C-3062) and incubated for 2hours at 37° C., followed by washing the wells three times with PBST.The plate was incubated with 100 μl/well of 1:50,000 dilution ofalkaline phosphatase-conjugated anti-rabbit IgG (Sigma A-2556) for 2hours at 37° C. and washed three times with PBST. The plate was finallyincubated with 100 μl/well of Lumi-Phos® Plus (Lumigen, Inc. P-701) for30 minutes at 37° C. and the enzyme-substrate reaction was measured in aMicrolite™ ML3000 Microtiter® Plate Luminometer (Dynatech Laboratories).

In the chemiluminescent G_(M1)-ELISA method, the amount of plant CTBfusion protein was measured by comparison of chemiluminescentintensities from a known amount of bacterial CTB protein-antibodycomplex with that emitted from a known amount of transformed plantsoluble protein. Two standard curves (1% and 0.1%) were generated basedon the relative light units (RLU) measured for different amount ofbacterial CTB. The RLU generated from serial dilutions of transgenicpotato plant homogenates were plotted into the graph, and found toreside within the 0.1% and 0.01% curves, indicating that the fusionprotein level in the transgenic potato tissue is slightly less than0.1%.

In the chemiluminescent immunoblot method, luminescent intensities ofbacterial and plant CTB protein bands blotted on Immun-Lite membranesafter SDS-PAGE were measured by the Argus-100 low-light imager DataAnalysis Program. The number of photons emitted from either bacterialCTB or plant CTB or plant CTB-NSP4 fusion protein bands were quantified,and their values compared to provide a semi-quantitative estimate of theamount of plant synthesized CTB fusion protein. Based on the amount oflight emission detected from a known amount of bacterial CTB protein(100 ng), the amount of plant CTB fusion protein was calculated to beapproximately 100 ng. The percent of chimeric protein in the plant wascalculated based on the amount of soluble plant protein (100 μg) used inthe assay. Based on this method, the percent of plant CTB protein wasfound to be approximately 0.1% of total soluble plant protein, a valuein close agreement with measurements made by the chemiluminescentG_(M1)-ELISA method. Based on the results of the chemiluminescent ELISAand immunoblot assays, 1 g of callus tissues (fresh weight) obtainedfrom auxin-induced potato leaves contained 10 μg of recombinant plantCTB-NSP4 fusion protein.

Pentamerization of CTB subunits is essential for its affinity for thenatural receptor. In G_(M1)-ELISA binding assays, plant-producedchimeric protein and bacterial CTB demonstrated a strong affinity forG_(M1)-ganglioside but not for BSA, which was the bases of proteinproduction level measurement. The ability of plant-derived CTB to bindG_(M1)-ganglioside indicates that the specific protein-gangliosidebinding interactions between amino acid residues forming the G_(M1)binding sites and the oligosaccharide moiety of G_(M1)-ganglioside areconserved. The strong binding efficiency of plant CTB conjugate forG_(M1) indicate that molecular configurations of CTB moiety is wellconserved. In addition, the absence of a monomeric form of chimera byimmunoblot analysis indicates that predominant molecular species ofchimeric protein is in the pentameric form, because monomeric CTB isunable to bind to G_(M1)-ganglioside. Therefore, the monomeric B subunitfusion polypeptide accumulated within the lumen of the ER of plant cellsand self-assembly into pentameric G_(M1) binding forms took place.

-   2) Method of Construction of a Transgenic Plant Producing a Fusion    Protein Comprising the Immunodominant Epitope of the Murine    Rotavirus Enterotoxin NSP4 Fused to the Cholera Toxin B Subunit and    the ETEC Fimbrial Antigen CFA/I Fused to the Cholera Toxin A2    Subunit and Confirmation of Transformation.

According to another embodiment of the present invention, there isprovided a transgenic plant producing a fusion protein comprising thetwenty-two amino acid immunodominant epitope of the murine rotavirusenterotoxin NSP4 fused to the cholera toxin B subunit and the ETECfimbrial antigen CFA/I fused to the cholera toxin A2 subunit. Theimmunodominant epitope of the murine rotavirus enterotoxin NSP4, thecholera toxin B subunit and the ETEC fimbrial antigen CFA/I function asantigens. The cholera toxin B subunit functions as an antigen and as anadjuvant. The cholera toxin A2 subunit functions as an adjuvant. Thetransgenic plant can be administered to a mammal to immunize the mammalagainst cholera, rotavirus and enterotoxigenic E. coli infectionsimultaneously.

As disclosed in greater detail below, the cholera toxin fusion proteinsexpressed in transformed potato tuber tissues assembled into a choleraholo-toxin-like oligomeric structure, which retained enterocyte membranereceptor G_(M1)-ganglioside binding affinity. Both serum and intestinalantibodies against NSP4, CFA/I and CTB were induced in orally immunizedmice. Analysis of IL-2, IL-4 and INFg cytokine levels in spleen cellsisolated from immunized mice indicated the presence of a strong Th1immune response to the plant synthesized antigens. Fluorescent antibodybased cell sorting (FACS) analysis of immunized mouse spleen cellsshowed an increase in CD4⁺ but not CD8⁺ memory cell populations.Following rotavirus challenge, passively immunized mouse pups showed a50% reduction of diarrhea symptoms.

Referring now to FIG. 2, there is shown a diagram of the vector used toprepare the transgenic plant. As can be seen, the vectorpPCV701CFA/I-CTB-NSP4 contained four genes located within thetransferred DNA (T-DNA) sequence flanked by the right and left border(RB and LB), and 25 bp direct repeats required for integration of theT-DNA into plant genomic DNA. The four genes were theCTBH:NSP4(114-135):SEKDEL coding sequence, SEQ ID NO:11, under controlof the mas P2 promoter; the CFA/I:CTA2 (SEQ ID NO:12 and SEQ ID NO:13)coding sequence under control of the mas P1 promoter; an NPT IIexpression cassette in the T-DNA to provide resistance to kanamycin inplants for selection of transformed plants; and a β-lactamase cassettefor resistance to ampicillin in E. coli and carbenicillin in A.tumefaciens. The g7pA polyadenylation signal was from the A. tumefaciensT_(L)-DNA gene 7. The OcspA polyadenylation signal is from the octopinesynthase gene. Each cholera toxin fusion gene contains its own leadersequence and an ER retention signal. To increase the flexibility of thefusion protein, a four amino acid glycine-proline (GPGP) hinge regionwas inserted between the CTB and NSP4 peptides.

The expression vector pPCV701CFA/I-CTB-NSP4 was assembled from theparental plasmid pPCV701 in the following manner. A nucleotide sequenceencoding the endoplasmic reticulum (ER) retention signal, SEKDEL, wasfirst cloned into the plant expression vector pPCV701on the P2 site ofthe mannopine synthase (mas) dual P1, P2 promoter. The CTB gene, SEQ IDNO:1, were amplified by polymerase chain reaction (PCR) from the choleratoxin (ctxAB) operon in plasmid pPT42. The CTB 3′ primer, SEQ ID NO:3,was designed to contain an oligonucleotide encoding the tetrapeptidehinge (Gly-Pro-Gly-Pro) to incorporate a degree of flexibility betweenthe CTB and NSP4 peptides. A synthesized DNA fragment, SEQ ID NO:8,encoding the rotavirus enterotoxin NSP4 (114-135), epitope was insertedin frame between the CTB-hinge and the SEKDEL sequences. The CTA leadersequence, SEQ ID NO:14, and the CTA2 gene were amplified by PCR from thectxAB operon and cloned into pPCv701 downstream of the mas P1 promoterregion. A DNA fragment, (431 bp), SEQ ID NO:12, encoding theenterotoxigenic E. coli colonization factor CFA/I, was amplified fromplasmid pIGx15A, and was inserted in frame between the CTA leadersequence, SEQ ID NO:14, and the CTA2 gene, SEQ ID NO:13. The whole CTAleader-CFA/I-CTA2 fusion gene is given as SEQ ID NO:15.

The resultant plant expression vector pPCV701CFA/I-CTB-NSP4, wasintroduced into Agrobacterium tumefaciens strain GV3101 pMP90RK. Fromsterile plants grown in culture medium in a light room, potato (Solanumtuberosum cv. Bintje) leaf tissue explants were transformed with A.tumefaciens harboring the plant expression vector pPCV701CFA/I-CTB-NSP4. Transformed plants were regenerated from the explants onselection medium containing kanamycin. Prior to analysis of antigen geneexpression, transgenic tubers were stimulated to produce high levels ofthe antigen proteins by incubation of tuber slices on growth mediumcontaining auxin 2,4-D (2,4 dichlorophenoxy acetic acid) for 4 days atroom temperature.

The presence of the CFA/I and CTB-NSP4 fusion proteins in thetransformed plants were detected by immunoblot techniques as follows.Protein extracts from auxin stimulated transformed potato tuberscontaining 100 mg of total soluble protein (TSP) were loaded on a 10-15%SDS-PAGE gel with or without 5 minutes boiling prior to electrophoresis.The separated protein bands were transferred to nitrocellulose membraneby electroblotting on a semi-dry blotter (Sigma) at 30V, 60 mA for oneand a half hours. The location of CTB, NSP4 and CFA/I proteins wereidentified by incubation of the blot in rabbit anti-CTB antiserum (Sigma1:5000 dilution) overnight at room temperature followed by incubation inalkaline phosphatase-conjugated mouse anti-rabbit IgG (Sigma, at1:10,000 dilution) for 2 hours at room temperature. Finally the membranewas incubated in the substrate BCIP/NPT (Sigma) for 10 min. The colorreaction was stopped by washing the membrane several times in distilledwater.

The bacterial CTB assembled into an oligomeric structure with amolecular weight of 45 kD, characteristic of the CTB pentamer. Thetransgenic plant produced CTB-NSP4 fusion peptide formed a 50 kDaoligomeric structure. The 5 kDa increase in molecular mass is consistentwith the presence of the additional NSP4 peptide and the 6 amino acidSEKDEL signal. The plant sample containing both CFA/I-CTA2 and CTB-NSP4fusion proteins showed the presence of a 70 kDa protein band, indicativeof the insertion of CFA/I-CTA2 peptide into the CTB-NSP4 pentamer. Theuntransformed plant showed no cross reaction with the cholera toxinantibody. Immersion of the samples in boiling water for 5 minutesresulted in dissociation of the multimeric structures into monomers. Thebacterial CTB monomer has a molecular mass of 11 kDa. The plant derivedCTB-NSP4 multimer dissociated into an 18 kDa monomer which is consistentwith the molecular mass of CTB plus NSP4.

-   3) Method of Immunizing a Mammal Against Infectious Diseases and    Analysis of the Results.

A group of 10 CD-1 female mice each were fed 3 g transgenic potato tubertissues containing a total of 7 mg of the recombinant fusion proteinspreviously determined by chemiluminescent ELISA on day 0, 5, 15, 23 and56. Using the same feeding schedule, a group of 5 CD-1 mice each werefed 3 g of untransformed potato tuber tissues as a negative control. Toevaluate the adjuvant effect of the CTB protein in the CTB-NSP4 fusion,CD1 mice (5 per group) were gavaged with pure NSP4 peptide with orwithout pure bacterial CTB (adjuvant) according to the same oralinoculation schedule. On day 13 after the final immunization, blood wastaken from each mouse for serum antibody titer determination. Three miceper group were euthanized at three different time points: 13, 34 and 68days after the fifth immunization. Intestinal washings were collectedfor mucosal antibody detection. Spleen cells from both immunized andnegative control mice (3×10⁶ cells/well) were suspended in RPMI 1640medium containing 10% fetal calf serum in duplicate samples, in 24 welltissue culture plates. After incubation for 72 hours at 37° C. in ahumidified, 5% CO₂ incubator, supernatants from the spleen cell cultureswere collected for assessment of IL-2, IL-4 and INFg secretion.

Following the five oral inoculations with transgenic potato tubertissues, blood samples were collected and the serum anti CTB, NSP4 andCFA/I IgG titers were measured by ELISA methods used in our laboratory.Out of 10 mice, 8 generated serum IgG against CTB with a mean titer of312.5±81.3. Of the 10 immunized mice, 8 developed serum IgG against NSP4with a mean titer of 125±61.23. Out of the 10 immunized mice, 10developed serum IgG against CFA/I with a mean titer of 84±44.2.

Intestinal IgG and IgA antibody titers against the three antigens wereanalyzed by chemiluminescent ELISA method used in our laboratory. Out of10 immunized mice, 5 generated measurable intestinal anti-CTB antibodytiters; 5 were found to have measurable intestinal anti-NSP4 antibodytiters and 6 were found to have significant intestinal anti-CFA/Iantibody titers. Negative control mice fed untransformed potato tubertissues did not develop detectable specific serum or mucosal antibodies.Since the CTB pentamer can bind to G_(M1) ganglioside located on themucosal epithelial cell surface, induction of both systemic and mucosalantibodies in the immunized mice indicated the successful delivery ofthe cholera toxin fusion proteins to the GALT.

Adjuvant and carrier functions of CTB in the CTB-NSP4 fusion proteinwere determined by measuring serum anti-NSP4 antibody titers in micefrom different vaccination groups. Mice fed the NSP4 peptide alonegenerated the lowest anti-NSP4 titer. Immunization with 7 mg ofbacterial CTB (the same amount detected in the plant derived CTB-NSP4fusion protein) increased the serum anti-NSP4 IgG titer approximatelytwo fold. Mice fed 3 g transformed potato tuber tissues containing theCTB-NSP4 fusion protein developed the highest anti-NSP4 titer.

Small soluble proteins like the NSP4 22 amino acid epitope that arehighly imunogenic by parenteral routes are frequently ineffective whenadministered orally unless a large dose of the protein is used. Thisresult can be attributed to intestinal digestion and lack of tropism ofthe peptide for the gut associated lymphoid tissues. Either choleraholotoxin or the CTB subunit, which function as mucosal adjuvants canstimulate an immune response against co-administered protein antigens.Directly linking small antigens with CTB subunit not only results inspecific targeting of the antigens to the mucosal immune system viaspecific enterocyte attachment but also increases the local antigenconcentration at the mucosal surface, which may explain our detection ofthe strongest immune response directed against the CTB-NSP4 fusionprotein.

The T lymphocyte populations in immunized mice were analyzed inimmunized mice as follows. IL-2, IL-4 and INFg produced in the spleencell culture supernatants were assayed by ELISA. Spleen lymphocytes werestained with fluorochrome-labeled monoclonal antibodies (mAb) forimmunophenotyping. Two monoclonal antibody panels were constructed forthree color analysis (fluoresceinisothiocanate (FITC), phycoerythrin(PE), and Cy-Chrome).

The first combination used, CD62L*FITC/CD4*PE/CD44*Cy-Chrome designatesnaïve and memory T helper cells. The second combination,CD62L*FITC/CD8b.2*PE/CD44*Cy-Chrome designates naïve and memorycytotoxic T cells. The spleen cells were resuspended at 10⁶ cells/ml inPBS and stained with fluorochrome-labeled mAbs. The labeled cells wereanalyzed by fluorescene activated cell sorting (FACS) to determine the Tlymphocyte memory cell sub-populations.

Following multiple oral immunizations, the Il-2 and the INFg expressionlevels in spleen cells dramatically increased, reaching the highestlevel 34 days after the fifth immunization and decreasing to basallevels by 68 days after vaccination. Throughout this time period IL-4levels remained low equivalent to that found in unimmunized mice. Thus,a cytokine expression pattern clearly indicated a Th1 lymphocytemediated immune response generated by feeding mice the plant derivedcholera toxin fusion antigens. Therefore, the overall cytokine secretionpattern of this multicomponent plant vaccine indicates a strong Th1response. FACS analysis of spleen cells collected on day 13, 34 and 68after the last immunization showed an elevated population of CD4⁺ memorycells in comparison with the unimmunized mice through the two monthsafter immunization. The CD4⁺ memory cell subpopulation (CD62⁻ CD44⁺,gate R4) detected in the immunized mice was observed to be significantlyhigher than the CD4⁺ memory cell subset in unimmunized mice. Thus, thegeneration of a significantly increased T helper memory cell populationin the immunized mice indicated successful protective immunizationmediated by the plant delivered antigens. The existence of increasednumbers of memory cells provided the ability to mount a strong immuneresponse following a second encounter with the same pathogen. The CD8⁺memory cell population detected in immunized mice did not show anysignificant increase over the unimmunized mouse negative control group.

Protection against rotavirus was evaluated as follows. Adult female CD-1mice (five per group) were fed 3 g of untransformed or transgenic potatotuber slices once a week for four weeks. Immediately following thefourth immunization at maximum anti-NSP4 antibody titer, the mice weremated with uninfected males. After a 19-20 day gestation period, mousepups were born to the immunized dams. On day 6 post parturition, eachpup received one oral dose of simian rotavirus SA-11 in 50 ul PBS thatcontained 15 DD₅₀ (the virus dose determined empirically to causediarrhea in 50% of the mouse pups). The mice were examined for thepresence of diarrhea daily for 5 days following inoculation by gentlepalpation of their abdomen to produce fecal pellets. The diarrhea scoreand the proportion of mice showing diarrhea symptoms in each study groupwere recorded.

The number of pups which developed diarrhea symptoms and the duration ofthe diarrhea was significantly reduced in the pups passively immunizedwith CTB-NSP4 fusion protein in comparison with pups born to unimmunizeddams. On day 3 after rotavirus challenge, a 50% reduction of diarrheasymptoms was detected in the immunized pups. Complete resolution ofdiarrhea symptoms occurred 4 days after virus challenge in pups fromimmunized dams. To exclude the possibility of diarrhea reduction due tothe presence of anti-CTB antibodies, pups born to dams immunized withplant derived CTB only were also challenged with an identical dose ofrotavirus SA11. No reduction of diarrhea symptoms was detected in miceimmunized with plant derived CTB alone. This experiment demonstratedthat anti-NSP4 antibodies generated in orally immunized mice were passedon to the pups and protected them from the onset of rotavirus infectionas well as significantly reducing the duration of the virus infection.

Therefore, according to one embodiment of the present invention, thereis provided a method of inducing partial or complete immunity to aninfectious disease in a mammal. The method comprises providing to themammal for oral consumption an effective amount of a fusion proteinaccording to the present invention. Preferably, the fusion protein ismade in a transgenic plant. Further preferably, the fusion proteincomprises a multimeric a cholera toxin B subunit and a first immunogenicantigen from a causal factor of the disease. In a preferred embodiment,the fusion protein additionally comprises a second immunogenic antigenfrom a causal factor of a mammalian disease fused to a cholera toxinsubunit, such as cholera toxin subunit A2. The cholera toxin subunitsact as adjuvants for the immunogenic antigens and, in the case ofcholera toxin B subunit, also act as an immunogenic antigen againstcholera infection.

The fusion protein can be provided to the mammal in a dose and frequencysufficient to render the mammal partially or completely immune from thefirst infectious disease, the second infection disease, cholera or acombination of the preceding. The specific dose and frequency aredetermined by well-known techniques as will be understood by those withskill in the art with reference to this disclosure.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

1. A method of inducing partial or complete immunity to gastroenteritisin a mammal comprising providing to the mammal for oral consumption aneffective amount of a protein complex comprising five monomeric fusionproteins; where each fusion protein comprises a cholera toxin B subunitlinked to NSP4 antigen from rotavirus; and where the protein complexfurther comprises ETEC fimbrial antigen CFA/1 from enterotoxigenic E.coli.
 2. A method for the prevention of multiple types of acutegastroenteritis simultaneously in a mammal comprising providing to themammal for oral consumption an effective amount of a protein complexencoded by a DNA construct that encodes a protein complex comprisingfive monomeric fusion proteins when expressed in a plant cell; whereeach fusion protein comprises a cholera toxin B subunit linked to NSP4antigen from rotavirus; and where the protein complex further comprisesETEC fimbrial antigen CFA/1 from enterotoxigenic E. coli.
 3. The methodof claim 2, where the DNA construct comprises a bidirectional promotercontrolling transcription of the DNA construct.